Bacterial expression of the shrimp molt-inhibiting hormone (MIH): antibodyproduction, immunocytochemical study and biological assay

Molting in shrimp is controlled by the molt-inhibiting hormone (MIH) and ec dysone. MIH inhibits the synthesis of ecdysone in the Y-organ, resulting in molt suppression; it is a neuropeptide member belonging to the eyestalk CH H/MIH/GIH family. The cloning of MIH (formerly MIM-like) of the shrimp Meta penaeus ensis has been reported in a previous study. To obtain a large quan tity of fusion protein for antibody production and biological assay, the cD NA encoding the shrimp MIH was inserted into the pRSET bacterial expression vector. His-tagged fusion protein was produced and purified by an Ni2+-cha rged affinity column. Polyclonal antibody to rMIH was subsequently obtained by immunizing rabbits with purified recombinant proteins. Results from Wes tern blot analysis indicated that the antibody was specific. Furthermore, r esults from immunocytochemical analysis showed that specific cells in three different clusters of the X-organ, the sinus gland and the axonal tract of the eyestalk contain MIH. To test for the molt-inhibiting activity of rMIH , shrimp at intermolt stage were injected with rMIH and the molt cycle dura tion of the injected shrimp was monitored. A significant increase in molt c ycle duration was recorded for the shrimp injected with the recombinant pro tein.